Mitochondrial dysfunction and disruption of the electron transport chain (ETC) has been identified as an important factor in diseases ranging from neurodegenerative diseases including, Alzheimer's disease (AD), Parkinson's disease (PD) and Friedreich's ataxia (FRDA), to diseases of the cardiovascular system, cancer and diabetes (Armstrong, J. S. et al. FASEB J. 2010, 24, 2152; Markesbery, W. R. et al. Brain Pathol. 1999, 9, 133; Barnham, K. J.; Masters, C. L. et al. Nat. Rev Drug Discovery. 2004, 3, 205; Calabrese, V. et al. J Neurol Sci. 2005, 233, 145; and Lin, M. T. et al. Nature 2006, 443, 787). The broad impact of mitochondria in so many diseases makes them prime targets for therapeutics. As powerhouses in mammalian cells, mitochondria are responsible for the predominant mode of energy production via oxidative phosphorylation (OXPHOS) of glucose, which is performed by the four respiratory complexes (complexes I-IV) and the ATP synthase (complex V), all located in the inner mitochondrial membrane (Henze, K. et al. Nature 2003, 426, 127; Saraste, M. W. Science 1999, 283, 1488; Newmeyer, D. D. et al. S. Cell 2003, 112, 481; and Fiore, C. et al. Biochimie 1998, 80, 137). However, mitochondria are also the major sites for production of reactive oxygen species (ROS) (Turrens, J. F. J. Physiol. 2003, 552, 335; and Murphy, M. P. Biochem. J. 2009, 417, 1). The impaired oxidative phosphorylation function would lead to further production of ROS, which further overwhelms the endogenous antioxidant systems and exposing cellular macromolecules to oxidative damage (Mates, J. M. et al. Clin Biochem. 1999, 32, 595; and Gaetani, G. F. et al. Blood 1989, 73, 334). Giving the main role of mitochondrial dysfunction in the development of several metabolic disorders, extensive research has explored therapeutic strategies for preserving mitochondrial function and the treatment of mitochondrial and neurological diseases (Heller, G. et al. Eur. J. Pharm. Biopharm. 2012, 82, 1; Smith, R. A. et al. Trends in Pharmacol. Sci. 2012, 341; Frantz, M. C. et al. Environ. Mol. Mutagen 2010, 51, 462; Arce, P. M. et al. ACS Med. Chem. Lett. 2011, 2, 608; Atamna, H. et al. FASEB. J. 2008, 22, 703; and Wen, Y. et al. J. Biol. Chem. 2011, 286, 16504).
Methylene blue (MB), a member of the phenothiazine family, originally discovered as a synthetic cationic dye. MB has a long-standing, extensive history of medical uses for more than a century (Wainwright, M. et al. J Chemother. 2002, 14, 431). It is an FDA approved drug for methemoglobinemia and an antidote to ifosfamide-induced encephalopathy (Wright, R. et al. Ann. Emerg. Med. 1999, 34, 646; and Zulian, G. B. et al. N. Engl J Med. 1995, 332, 1239). MB was also the lead compound for successful pharmacotherapeutic derivatives such as the antimalarial agents such as quinacrine, and chloroquine (Green, R. Lancet 1932, 219, 826; and Loeb, R. F. et al. J. Am. Med. Assoc. 1946, 130, 1069).
MB has very unique redox property that exists in equilibrium between oxidized state in dark blue and colorless reduced state, making it both antioxidant and prooxidant under different conditions. MB with a mild redox potential, appears to readily cycle between the oxidized and reduced forms using specific mitochondrial and cytosolic redox centers. This property of MB has been reported to redirect and facilitate electron transfer across mitochondrial electron transfer complexes minimizing electron leakage, and inhibit superoxide production (mitochondrial electron-carrier bypass) (Wen, Y. et al. J. Biol. Chem. 2011, 286, 16504; and Wainwright, M. et al. J Chemother. 2002, 14, 431). Thus, MB might be able to act as an alternative electron transfer carrier that replaces the damaged mitochondrial respiratory chain.
Methylene blue has limitation in term of bioavailability and tolerability (at high doses). The substantial hydrophilicity of MB may restrict its permeability across the plasma membranes of mammalian cells, which would also limit its cellular uptake. It is for this reason TauRx switch to the reduced form of methylene blue (more hydrophobic) in their phase III clinical trials (Baddeley, T. C. et al. J Pharmacol Exp Ther. 2015, 352, 1; and Melis, V. et al. Behav Pharmacol. 2015, 26, 353). MB has a hormetic dose-response in which its beneficial effects are optimal in the lower to intermediate range (Bruchey A. K. et al. Am J Pharm Toxicol 2008, 3, 72). Previous study has shown the effect and interaction of exogenous short-chain CoQ10 analogues on mitochondrial oxidative phosphorylation in isolated mitochondria and ROS metabolism in cultured cells. It was clearly showed that the antioxidant reactions of exogenous ubiquinones will predominantly occur within phospholipid bilayers, while the pro-oxidant reactions require an aqueous environment. It is apparent that increasing hydrophobicity limits the “bad” aqueous autoxidation of ubiquinols without diminishing its “good lipid phase antioxidant functions. This could provide explanation for the extreme hydrophobicity of endogenous CoQo10. Therefore, the relative rates of these reactions can be fine-tuned by hydrophobicity, allowing a rational approach to the design of therapeutic mitochondria-targeted redox cyclers.
Methylene violet (MV) is a neutral phenothiazine dye. Hydrolysis of MB under strongly basic condition yields MV with improved hydrophobicity (Houghtaling, M. A. et al. Photochem. Photobiol. 2000, 71, 20). MV can be looked upon as a phenolic analogue of MB where a hydroxyl group substitutes one of the two dimethylamine moieties. Although MV is naturally obtained in the oxidized (quinone) form, it can be reduced by the mitochondrial redox centers generating the phenolic (quinol) form. The reduced form of MV can act as phenolic antioxidants similar to CoQ10. Currently there is a need for new antioxidant compounds with reduced cytotoxicity and side effects (e.g. by decreasing the pro-oxidant effect).